Method for Analyzing the Antimicrobial Activity of Peptides via Escherichia coli Expression System

The test system for an assay of new potential antimicrobial peptides (AMP) based on the expression of recombinant AMP-encoding genes in Escherichia coli cells has been proposed. This method has a number of advantages over the use of chemically synthesized peptides and both approaches effectively complement each other. Our approach does not impose limitations on the AMP size, facilitates high-throughput screening of mutant plasmid libraries, and has lower cost and complexity compared to the use of synthetic peptides. The core of our methodology involves transformation of the model gram-negative bacterium E. coli with plasmids carrying a recombinant AMP-encoding gene regulated by an inducible promoter. Following transcription induction, bacteria synthesize the AMP, which ultimately leads to cell death. The assessment of bacterial growth is carried out either by measuring the optical density of a bacterial culture grown in liquid media in a microplate or by drip seeding of serial culture dilutions on an agar-based nutrient medium.